Background and Aims In recent years, the incidence of intrahepatic cholangiocarcinoma (ICC) has increased. Due to its insidious onset and atypical early symptoms, most ICC patients are diagnosed at a late stage, leading to a poor prognosis. ICC cell lines are valuable in vitro models widely used in basic and clinical research on ICC. In-depth studies and drug screening of these cell lines may improve patient outcomes. Increasing evidence suggests that the morphological structure of tumor cells significantly impacts tumor development, progression, and drug sensitivity. Only a limited number of ICC cell lines derived from Chinese patients have been established, and their characteristics, particularly their ultrastructural features, are not fully characterized. Therefore, this study investigated the morphological and ultrastructural characteristics of the ICC-X1 cell line, aiming to provide a reference for future research.Methods The newly established Chinese human ICC cell line ICC-X1 was cultured and processed. The microscopic structure, surface morphology, and ultrastructure of ICC-X1 cells, including the cell membrane, cytoplasm, and organelles, were observed and analyzed using light, scanning, and transmission electron microscopy, respectively.Results Under optical microscopy, ICC-X1 cells exhibited adherent growth, predominantly polygonal and short spindle-shaped morphology, with a high prevalence of mononuclear cells, large nuclei, and scant cytoplasm, resulting in an increased nucleocytoplasmic ratio. When adequately nourished, the cells showed layered growth and lost contact inhibition. Scanning electron microscopy revealed an abundance of microvilli surrounding the ICC-X1 cells, with uniform thickness and length and long pseudopodia. Transmission electron microscopy showed that ICC-X1 cells had giant, eccentrically placed nuclei with uneven electron density of chromatin, and abundant mitochondria and ribosomes were observed.Conclusion This study provides detailed data on the morphology and ultrastructure of the ICC-X1 cell line, offering insights and perspectives for exploring the relationship between ICC cell structure and function and investigating the mechanisms of ICC drug resistance.